This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In our grant, IR21AI074898, "A Primate Model of Mycoplasma genitalium" we will assess the ability of pigtail macaques to serve as animal models to study the immunopathogenesis of M. genitalium (MG), a newly recognized reproductive tract pathogen with a disease spectrum and significance that appears to be very similar to Chlamydia trachomatis. Thus, we will assess the ability of cervically-inoculated MG to infect, persist, and induce an innate and adaptive immune response in the ectocervix, endocervix, and vagina, and ascend into upper reproductive tract to elicit salpingitis. Further we will evaluate the occurrence of MG gene variation during infection and its effect on survival of this bacterium. In our initial experiments, we determined that MG isolates recovered from primates were inhibited for growth in the standard H broth used for in vitro growth of this fastidious organism. Subsequently we determined that this inhibition could be overcome by dilution of the primate specimen in H broth or by co-culture of the infected specimen with Vero cells. We are currently evaluating whether other Mycoplasma spp. colonizing the lower genital tract of these macaques mediate this inhibition. As a result, we have adapted our primate protocols to routinely include inoculating primate specimens to H agar plates (to independently assess the presence of MG and other Mycoplasma-like organisms), into Vero cell cultures, and into serial dilutions of H broth. Using reconstruction experiments with primate tissue (available through the WaNPRC necropsy program), we have also developed methods to improve homogenization of primate tissues to maximize the recovery of viable MG. We have also demonstrated that MG gene sequence variation consistent with antigenic variation of surface proteins occurs during primate infection but not in the same inoculum of MG cultivated concurrently in vitro, supporting a role of antigenic variation in persistence of MG in vivo.